Extraction methods for preparing thromboplastin reagents

ABSTRACT

The Prothrombin Time (PT) is used as a screening test for blood coagulation factor deficiencies and for monitoring oral anti-coagulant therapy using coumadin. Thromboplastin reagents activate the extrinsic pathway of coagulation and are the basis for the Prothrombin Time (PT) test. This invention describes the use of barium sulfate and chaotropic agents, and nonionic detergents, for the extraction of sensitive thromboplastin reagents from tissue. Extraction with sodium thiocyanate alone also greatly enhances thromboplastin sensitivity. This invention should be useful for all thromboplastins and will improve their sensitivity for all PT-based tests and specific assays.

This is a continuation of application Ser. No. 07/632,894, filed on Dec.24, 1990, now abandoned, which is a division of U.S. Ser. No. 07/276,083filed Nov. 23, 1988, now abandoned.

FIELD OF THE INVENTION

This invention relates to an improved method of extracting and preparingthromboplastin reagents.

BACKGROUND OF THE INVENTION

Thromboplastin reagents activate the extrinsic pathway of coagulationand are the basis for the prothrombin time (PT) test. The PT test isused to screen for blood coagulation factor deficiencies and formonitoring oral anticoagulant therapy (e.g. coumadin). Reagents for PTtests include tissue thromboplastin, also called tissue factor, andcalcium ions as active ingredients diluted with appropriate buffers andstabilizers. Thromboplastin forms a complex with coagulation factor VIIto greatly enhance its proteolytic activity.

Thromboplastin may be derived from a variety of tissues of differentanimal sources. Each tissue has a characteristic activity andsensitivity to coagulation enzymes (i.e., factors); these properties aremodulated by other constituents of the reagent. Thromboplastinsensitivity is defined as the prolongation of the clotting times of bothcoumadinized plasmas and plasmas deficient in clotting factors II, V,VII, and X. Sensitivity to coumadinized plasma is assessed by taking theratio of an abnormal plasma sample to a normal plasma sample.

Currently, the most sensitive thromboplastin reagents are derived fromhuman brain and placenta. The limited availability of these materials,their cost and the potential for HIV virus contamination limit theiruniversal acceptance. Thromboplastins derived from rabbit brain, themost common source, typically have relatively low sensitivity comparedto thromboplastins derived from human tissues. The source of thethromboplastin, the method of extracting the thromboplastin and thereagent composition are all important parameters in determining reagentsensitivity. Variations in the composition of PT reagents can also beused to improve stability and adjust clotting times of plasma samplesfrom normal individuals.

Historically thromboplastin has been extracted from tissues by heatingthe tissue in water or saline solutions. Thromboplatin reagents made byBaxter Healthcare Corporation, Dade Division, contain thromboplastinsextracted in saline-tartrate solutions (U.S. Pat. No. 3,522,148 - Jul.25, 1970). These extracts are centrifuged to remove large particles. Thesupernate thromboplastin extract contains the active thromboplastinalong with the sodium chloride and sodium tartrate from the extractionfluid. In Thromboplastin C the thromboplastin extract is added to asolution containing calcium lactate, sodium chloride, sodium tartrate,glycine and carboxymethyl cellulose. The final concentration of extractis 25% of the final reconstituted volume.

In Thromboplastin FS, the thromboplastin extract is added to a solutioncontaining imidazole, calcium lactate, sodium chloride, sodium tartrate,glycine and carboxymethyl cellulose. Because the final concentration ofextract is 50% of the final reconstituted volume, Thromboplastin FS(TPES) made by Baxter Healthcare Corporation, Dade Division, has arelatively high concentration of rabbit brain extract. While moresensitive than other rabbit brain thromboplastins, the normal range PTvalues are longer than desired, the turbidity is high and the stabilityis less than optimal.

Boehringer Mannheim has developed a process to make rabbit brainthromboplastin more sensitive (DE 3150594A1). In their procedure, rabbitbrain powder is mixed with equal parts of cellulose powder and washedwith sodium acetate buffer at pH 6.5-8 to remove contaminants such ashemoglobin. The brain residue is then extracted with surface activeagents, such as sodium deoxycholate in the presence of calcium-ions. Thekey constituent disclosed in the Boehringer Mannheim process is calciumions, and, if needed, a surface active agent is used. The use of bariumsulfate is discussed in the Boehringer Mannheim patent, but discounted:"according to our own experiences, the thromboplastin largelyco-precipitates with the barium sulfate." In conventional procedures therelative insensitivity of thromboplastins is due, in part, to thepresence of coagulation factor VII, being tightly bound to thethromboplastin. Barium sulfate is commonly used as an adsorbing agent toremove vitamin K-dependent coagulant proteins such as factors II, VII,IX, and X.

BRIEF SUMMARY OF THE INVENTION

The present invention uses various combinations of nonionic detergents,such as Triton® X-100, chaotropic ions such as thiocyanate, iodide,guanidine and perchlorate, and barium sulfate to extract thromboplastinfrom a tissue source. Nonionic detergents greatly enhance the efficiencyof removing the coagulation factors bound to the thromboplastin. Theapproach described herein offers the advantage of enhanced sensitivityto factor deficiencies and coumadin therapy while maintaining arelatively short normal range PT. A survey conducted by the CAPHematology Resource Committee in 1978 revealed that the normal range PTwas 9-15 seconds for 96% of the laboratories reporting (Triplett, D. A.,"How the Prothrombin Time Actually is Performed" in Standardization ofCoagulation Assays: An Overview, ed. by D. A. Triplett, College ofAmerican Pathologists, Skokie, 1982, pp. 113-119). The preferred PTrange for normal donors is 10-14 seconds. Reagents prepared using themethods described here have enhanced reconstituted stability overThromboplastin FS, use less brain extract and are less optically dense.The present invention allows sensitivity to Factor VII deficient plasmausing insensitive thromboplastin reagents to be set at any PT; thepreferred PT time for Factor VII deficient plasma is greater than sixtyseconds. The thromboplastin reagent of the present invention will have aratio of the abnormal control (COL2) to the normal control (COL1)greater than 1.8, however, preferably the ratio is in the range of 1.8to 3.0.

Alternatively, chaotropic ions, such as thiocyanate, guanidine, oriodide, used alone in the extraction fluid greatly enhance sensitivity.Chaotropic ions are agents used in disrupting membranes and enzymecomplexes by breaking noncovalent forces. (W. Hanstein, Destabilizationof Membranes with Chaotropic Ions, Meth. Enzy. XXXI (1974)). Chaotropicions typically have a low charge density with a large radius. Commonlyused ions include: tribromoacetate, trichloroacetate, guanidinium,thiocyanate, iodide, perchlorate, dichloroacetate, nitrate, bromide,trifluoroacetate and chloroacetate.

DETAILED DESCRIPTION AND BEST MODE

In this invention tissue, such as acetone-dehydrated rabbit brainpowder, is extracted with a combination of barium sulfate, nonionicdetergents and chaotropic ions, such as thiocyanate, iodide, guanidineor perchlorate, to control reagent properties. This invention should beuseful for all thromboplastins and will improve the sensitivity for allPT-based tests and specific assays.

PT values can be determined using automated coagulation analyzers, suchas the MLA Electra 800, mechanical instruments, such as Fibrometers, orby manual techniques. The abnormal plasma for these studies has beeneither an abnormal control such as Baxter Healthcare Corporation, DadeDivision, Ci-Trol® Level II (COL2) or lyophilized coumadinized plasmas(LAC) for an anticoagulant control. Ci-Trol® Level I (COLI) or a pool offresh normal citrated plasma (FNP) have been used to determine normal PTvalues. Sensitivity to factor deficiencies is assessed by measuring thePT of a factor VII-deficient plasma (CF7). Rabbit brain thromboplastinsavailable in the United States (Dade Thromboplastin C) yield ratios or1.5 for COL2/COL1 and a 28-30 sec PT for factor VII-deficient plasma onthe MLA Electra 800.

EXAMPLE I General Process Used to Prepare Thromboplastins

To make the thromboplastin reagent, rabbit brain powder (50 gm) isextracted in an extraction fluid (1000 mL) containing sodium chloride(30-180 mM), a nonionic detergent, such as Triton®X-100 (0.01-0.25%) anda chaotropic ion, such as sodium thiocyanate (5-100 mM). Alternatively,the extraction fluid may contain only sodium chloride (0-150 mM) and thechaotropic ion (5-100 mM). Barium sulfate powder is added to theextraction fluid at 0.1-1.0 gm/gm brain powder. The extraction isperformed at 43° to 47° C. for fifteen (15) minutes; the extractionmixture is then centrifuged for ten (10) minutes at 2500 RPM to removethe barium sulfate and large particles. The extract is added to a basecontaining calcium ions (7-14 mM), sodium chloride (70-150 mM), buffersand stabilizers. The product is then freeze-dried.

EXAMPLE II The Effect of Triton X-100 and Barium Sulfate in Extractionon Thromboplastin Sensitivity

Acetone-dehydrated rabbit brain powder (1 gm) was extracted in 20 mL of0.63% sodium chloride (NaCl) containing 0.6 gm of barium sulfate (BaSO₄)and 0-0.25% Triton X-100® (Rohm & Haas, Philadelphia, Pa.) for fifteen(15) minutes at 45° C.; the extraction mixture was then centrifuged at2500 RPM for ten (10) minutes to remove the barium sulfate and largeparticles. The extract was added to a base at a final concentration of32% in 30 mM (TAPSO) buffer, 5% glycine, 0.6% polyethylene glycol (PEG)with a molecular weight of 8000, 13.7 mM calcium chloride (CaCl₂), 100mM NaCl, pH 7.0. Prothrombin times (PT) in seconds were recorded usingan MLA Electra 800. (Medical Laboratory Automation, Pleasantville, N.Y.)

Both the ratio of COL2/COL1 and PT of factor VII-deficient plasma (CF7)increase substantially as the amount of detergent is increased (TableI).

                  TABLE I                                                         ______________________________________                                        Effect of Triton X-100 and Barium Sulfate in Extraction on                    Thromboplastin-bulk (MLA-800): Average of 2 replicates                                                    RATIO                                             % Triton X-100                                                                           COL 1   COL 2    COL 2/COL/1                                                                             CF7                                     ______________________________________                                        0% Control 11.8    20.6     1.75      95.9                                    0.05%      12.1    22.1     1.83      107.4                                   0.1%       13.0    25.1     1.93      120.4                                   0.25%      14.3    34.4     2.41      193.4                                   ______________________________________                                         Base Formulation:                                                             30 mM TAPSO, 5% Glycine, 0.6% PEG, 13.7 mM CaCl.sub.2, 100 mM NaCl, 32%       Extract, pH 7.0                                                               Brain extracted in 0.63% NaCl and detergent with barium sulfate at 0.6        gm/gm brain in 20 mL of extraction fluid                                 

EXAMPLE III The Effect of Sodium Thiocyanate (NaSCN) in Extraction onThromboplastin Sensitivity

Acetone-dehydrated rabbit brain powder (1 gm) was extracted in 20 mL of0.63% sodium chloride (NaCl), except where noted, containing 0(control), 10, 50, and 100 mM NaSCN for fifteen (15) minutes at 45° C.;a sample containing 100 mM NaSCN, without NaCl was also evaluated.Barium sulfate was not used in this experiment. The extraction mixturewas centrifuged as described in Example I. Prothrombin times (PT) inseconds were recorded using an MLA Electra 800.

Both the ratio of COL2/COL1 and PT of factor VII-deficient plasma (CF7)increase substantially as the amount of NaSCN is increased (Table II).Sodium thiocyanate alone increased thromboplastin sensitivity.

                  TABLE II                                                        ______________________________________                                        Effect of Sodium Thiocyanate (NaSCN) in Extraction on                         Thromboplastin - bulk (MLA-800)                                                                           RATIO                                             P2H         COL 1   COL 2   COL 2/COL 1                                                                              CF7                                    ______________________________________                                        CONTROL     11.9    21.5    1.81       37.3                                   10 mM NaSCN 12.5    24.1    1.93       67.4                                   50 mM NaSCN 13.7    30.9    2.26       159.3                                  100 mM NaSCN                                                                              14.7    35.6    2.42       177.7                                  100 mM NaSCN-                                                                             14.1    31.6    2.24       131.6                                  NO NaCl                                                                       ______________________________________                                         Base Formulation:                                                             30 mM TAPSO, 5% Glycine, 0.6% PEG, 13.7 mM CaCl.sub.2, 100 mM NaCl, 32%       Extract, pH 7.0                                                          

EXAMPLE IV Comparison of Various Compositions of Extraction Fluids andPercentage of Extract on Thromboplastin Sensitivity

Rabbit brain powder was extracted in extraction fluids containing two(2) different compositions of NaCl, Triton X-100, NaSCN and bariumsulfate. In Table III, Extract E contained 130 mM NaCl, 50 mM NaSCN,0.05% Triton X-100 and 0.3 gm barium sulfate/gm brain powder; Extract Ncontained 50 mM NaCl, 10 mM NaSCN, 0.02% Triton X-100 and 0.4 gm bariumsulfate/gm brain powder. The brain powder was extracted, centrifuged andthe extracts were added to a base to the final concentrations of 32% or50% as described in Example I. Prothrombin Times (PT) in seconds wererecorded using an MLA Electra 800.

Table III shows that the components of the extraction fluids and theamount of extract can vary considerably to yield thromboplastins withenhanced sensitivity over rabbit brain thromboplastins, such as DadeThromboplastin C (ratio COL2/COL1=1.5).

                                      TABLE III                                   __________________________________________________________________________    Comparison of Various Compositions of Extraction Fluids and                   Percentage of Extract on Thromboplastin - Bulk (MLA-800)                                        RATIO         RATIO                                         % Extract COL 1                                                                             COL 2                                                                             COL 2/COL 1                                                                           FNP                                                                              LAC                                                                              LAC/FNP                                                                             CF7                                     __________________________________________________________________________    32% Extract E                                                                           14.6                                                                              32.8                                                                              2.25    14.2                                                                             35.5                                                                             2.50  81.1                                    50% Extract E                                                                           16.9                                                                              45.5                                                                              2.69    15.8                                                                             50.4                                                                             3.19  155.1                                   32% Extract N                                                                           12.8                                                                              24.1                                                                              1.88    12.4                                                                             27.4                                                                             2.23  106.9                                   50% Extract N                                                                           13.9                                                                              32.3                                                                              2.32    13.4                                                                             39.0                                                                             2.91  176.6                                   Thromboplastin FS                                                                       13.3                                                                              24.5                                                                              1.84    13.0                                                                             31.4                                                                             2.42  64.6                                    __________________________________________________________________________     Base Formulation:                                                             30 mM TAPSO, 5% Glycine, 0.6% PEG, 13.7 mM CaCl.sub.2, 100 mM NaCl, pH 7.     Extract E:                                                                    Brain Powder extracted in 130 mM NaCl, 50 mM NaSCN, 0.05% Triton X100, 0.     gm barium sulfate/gm brain powder                                             Extract N:                                                                    Brain Powder extracted in 50 mM NaCl, 10 mM NaSCN, 0.02% Triton X100, 0.4     gm barium sulfate/gm brain powder                                        

EXAMPLE V Comparison of Various Formulations of SensitiveThromboplastins

Acetone-dehydrated rabbit brain powder was extracted in a solutioncontaining 50 mm NaCl, 10 mM NaSCN, 0.02% Triton X-100 and 0.4 gm bariumsulfate/gm brain powder. The brain powder was extracted as described inExample I. The extracts were added to bases for two (2) differentformulations: Formulation D contained 35% extract in 40 mm bicinebuffer, 5.25% glycine, 0.6% PEG, 10 mM CaCl₂, 134 mM NaCl, pH 7.1:Formulation T contained 36% extract in 80 mM TAPSO, 5.25% glycine, 0.6%PEG, 10 mM CaCl₂, 118 mM NaCl, pH 7.4. Both formulations werelyophilized. After reconstitution, PT in seconds was recorded using anMLA Electra 800.

Table IV shows that the composition of the formulation can varyconsiderably to yield thromboplastins with enhanced sensitivity andother properties superior to Thromboplastin FS, a relatively sensitiverabbit brain thromboplastin.

                                      TABLE IV                                    __________________________________________________________________________    Comparison of Various Formulations of Sensitive Thromboplastins - Bulk        (MLA-800)                                                                                       RATIO         RATIO                                         Formulation                                                                             COL 1                                                                             COL 2                                                                             COL 2/COL 1                                                                           FNP                                                                              LAC                                                                              LAC/FNP                                                                             CF7                                     __________________________________________________________________________    D         13.4                                                                              31.1                                                                              2.32    12.4                                                                             33.5                                                                             2.70  86.3                                    T         14.0                                                                              35.0                                                                              2.50    13.0                                                                             33.9                                                                             2.61  93.0                                    Thromboplastin FS                                                                       14.2                                                                              26.7                                                                              1.88    13.4                                                                             30.5                                                                             2.28  58.9                                    __________________________________________________________________________     Extract Mixture:                                                              Brain Powder extracted in 50 mM NaCl, 10 mM NaSCN, 0.02% Triton X100, 0.4     gm barium sulfate/gm brain powder                                             Formulation D:                                                                40 mM Bicine, 5.25% Glycine, 0.6% PEG, 10 mM CaCl.sub.2, 134 mM NaCl, pH      7.1                                                                           Formulation T:                                                                80 mM TAPSO, 5.25% Glycine, 0.6% PEG, 10 mM CaCl.sub.2, 118 mM NaCl, pH       7.4                                                                      

EXAMPLE VI Preparation of Thromboplastins with sensitivity typical ofthose sold in the U.S.

Acetone-dehydrated rabbit brain powder was extracted in solutioncontaining 50 mM NaCl, 10 mM NaSCN, 0.02% Triton X-100 and 0.4 gm bariumsulfate/gm brain powder. The brain powder was extracted as described inExample 1. The extracts were added to bases for three differentformulations: Formulation E contained 10% extract in 53 mM TAPSO buffer,4.00% glycine, 0.3% PEG, 11 mM CaCl₂, 50 mM NaCl, pH 7.4; Formulation Fcontained 12% extract in 50 mM TAPSO buffer, 4.00% glycine, 0.3% PEG, 11mM CaCl₂, 50 mM NaCl, pH 7.4; Formulation G contained 10% extract in 53mM TAPSO buffer, 4.00% glycine, 0.3% PEG, 11 mM CaCl₂, 65 mM NaCl, pH7.4. The formulations were lyophilized. After reconstitution, PT inseconds were records using an MLA Electra 800.

Table V shows that the extraction fluids described herein can be used tomake conventional rabbit brain thromboplastins available in the U.S.similar to Thromboplastin C. Relatively small amounts of extract (10-12%versus 25% for Thromboplastin C) are required to make a thromboplastinwith lower COL/COL1 ratio and LAC/FNP ratio. However, the sensitivity tofactor VII is enhanced.

                                      TABLE V                                     __________________________________________________________________________    Comparison of Various Formulations of Thromboplastins with                    Sensitivity Typical of those sold in the U.S. (MLA-800)                                         RATIO         RATIO                                         Formulation                                                                             COL 1                                                                             COL 2                                                                             COL 2/COL 1                                                                           FNP                                                                              LAC                                                                              LAC/FNP                                                                             CF7                                     __________________________________________________________________________    E         11.8                                                                              20.4                                                                              1.73    11.6                                                                             21.7                                                                             1.87  42.1                                    F         11.7                                                                              20.4                                                                              1.74    11.3                                                                             21.3                                                                             1.88  40.6                                    G         11.4                                                                              19.7                                                                              1.73    11.1                                                                             21.0                                                                             1.89  40.4                                    Thromboplastin C                                                                        11.9                                                                              20.2                                                                              1.70    11.5                                                                             20.2                                                                             1.76  27.7                                    __________________________________________________________________________     Extract Mixture:                                                              Brain Powder extracted in 50 mM NaCl, 10 mM NaSCN, 0.02% Triton X100, 0.4     gm barium sulfate/gm brain powder                                             Formulation E:                                                                10% Extract, 53 mM TAPSO, 4.00% Glycine, 0.3% PEG, 11 mM CaCl.sub.2, 50 m     NaCl, pH 7.4                                                                  Formulation F:                                                                12% Extract, 50 mM TAPSO, 4.00% Glycine, 0.3% PEG, 11 mM CaCl.sub.2, 50 m     NaCl, pH 7.4                                                                  Fomulation G:                                                                 12% Extract, 50 mM TAPSO, 4.00% Glycine, 0.3% PEG, 11 mM CaCl.sub.2, 65 m     NaCl, pH 7.4                                                             

Variants or Equivalents of the Invention

The performance and sensitivity of a PT reagent is the result ofinteractions between all of the constituents of the reagent. Generally,changes in formulation which increase the sensitivity also increase thevalue of the normal PT. In the present invention, the components of theextraction fluid are carefully balanced for best normal PT andsensitivity. In addition, the individual constituents of the extractionmixture influence reagent performance in specific ways. Varying theconcentrations of the various components also alters the properties ofthe prepared extract; therefore, extracts with different properties canbe prepared depending on the extraction fluid composition.

Barium sulfate and any nonionic detergent, such as Triton® X-100,Brij-35, Nonidet® P40, FSN® and Tergitol® greatly enhance sensitivity,especially to specific coagulation factor deficiencies such as factorVII. Chaotropic ions (thiocyanate, guanidine, iodide, perchlorate) alonein the extraction enhances sensitivity to both coumadinized patientsamples and specific factor deficiencies such as factor VII. To make thethromboplastin reagent, the tissue is extracted in an extraction fluidcontaining sodium chloride (30-180 mM), the nonionic detergent(0.01-0.25%) and the chaotropic ion such as thiocyanate, guanidine,iodide and perchlorate (5-100 mM). Barium sulfate powder is added to theextraction fluid at 0.1-1.0 gm/gm brain powder. Alternatively, theextraction fluid may contain only sodium chloride (0-150 mM) and sodiumthiocyanate (5-100 mM). The extraction is performed at 43° to 47° C. forfifteen (15) minutes; the extraction mixture is then centrifuged for ten(10) minutes at 2500 RPM to remove the barium sulfate and largeparticles. The extract is added to a base containing calcium ions (7-14mM), sodium chloride (70-150 mM), buffers and stabilizers. The buffersand stabilizers can be varied to improve the stability of the product aswell.

The extraction method and components can be used with any source oftissue containing thromboplastin, such as rabbit brain and lung, bovinebrain and lung, ovine brain and lung, and human brain, lung andplacenta.

What is claimed is:
 1. A diagnostic prothrombin time reagent comprisingan extracted thromboplastin composition said prothrombin time reagenthaving approximately normal prothrombin times and a sensitivity to bloodcoagulation factor VII deficiency of at least forty seconds at aconcentration of about 10% of said extracted thromboplastin compositionand sensitivity to coumadin therapy greater than 1.8, as measured by theratio of the prothrombin time value of an abnormal sample to a normalsample and wherein the extracted thromboplastin composition is made bythe process comprising:(a) contacting rabbit brain tissue containingthromboplastin with an effective amount of extraction fluid comprisingabout 0.1 to 1.0 grams of barium sulfate per gram of tissue, about 0.01to 0.25% of a nonionic detergent, about 5 to 100 mM chaotropic ion, anda salt, thereby extracting the thromboplastin into the fluid, and (b)separating said extracted thromboplastin from said depleted tissue andbarium sulfate.
 2. The diagnostic prothrombin time reagent of claim 1wherein the amount of extraction fluid is about 100 mL for every 5 gramsof tissue.
 3. The diagnostic prothrombin time reagent of claim 1 whereinthe concentration of the salt is from about 30 to 180 mM.
 4. Thediagnostic prothrombin time reagent of claim 1 wherein the normalprothrombin time ranges from about 9 to 15 seconds and the enhancedsensitivity to coumadin therapy shown by COL2/COL1 ratio ranges fromabout 1.8 to 3.0.
 5. A diagnostic prothrombin time reagent comprising anextracted thromboplastin composition said prothrombin time reagenthaving approximately normal prothrombin times and sensitivity to bloodcoagulation factor VII deficiency of at least sixty-five seconds at aconcentration of about 32% of said extracted thromboplastin compositionand sensitivity to coumadin therapy greater than 1.8, as measured by theratio of the prothrombin time value of an abnormal sample to a normalsample and wherein the extracted thromboplastin composition is made bythe process comprising:(a) contacting rabbit brain tissue containingthromboplastin with an effective amount of extraction fluid comprisingabout 5 to 100 mM chaotropic ions, thereby extracting the thromboplastininto the fluid, and (b) separating said extracted thromboplastin fromsaid depleted tissue.
 6. The diagnostic prothrombin time reagent ofclaim 5 wherein the amount of extraction fluid is about 100 mL for every5 grams of tissue.
 7. The diagnostic prothrombin time reagent of claim 5wherein the normal prothrombin time ranges from about 9 to 15 secondsand the enhanced sensitivity to coumadin therapy shown by COL2/COL1ratio ranges from about 1.8 to 3.0.
 8. A diagnostic prothrombin timereagent comprising an extracted thromboplastin composition saidprothrombin time reagent having approximately normal prothrombin timesand sensitivity to blood coagulation factor VII deficiency of at leastone hundred seconds at a concentration of about 32% of said extractedthromboplastin composition and sensitivity to coumadin therapy greaterthan 1.8, as measured by the ratio of the prothrombin time value of anabnormal sample to a normal sample and wherein the extractedthromboplastin composition is made by the process comprising:(a)contacting rabbit brain tissue containing thromboplastin with aneffective amount of extraction fluid comprising about 0.1 to 1.0 gramsof barium sulfate per gram of tissue, about 0.01 to 0.25% nonionicdetergents, and a salt, thereby extracting the thromboplastin into thefluid, and (b) separating said extracted thromboplastin from saiddepleted tissue and barium sulfate.
 9. The diagnostic prothrombin timereagent of claim 8 wherein the amount of extraction fluid is about 100mL for every 5 grams of tissue.
 10. The diagnostic prothrombin timereagent of claim 8 wherein the concentration of the salt is from about30 to 180 mM.
 11. The diagnostic prothrombin time reagent of claim 8wherein the normal prothrombin time ranges from about 9 to 15 secondsand the enhanced sensitivity to coumadin therapy shown by COL2/COL1ratio ranges from about 1.8 to 3.0.